Method, apparatus and kits for sequencing of nucleic acids using multiple dyes

ABSTRACT

An instrument for sequencing oligonucleotides is loaded with the products of four sequencing reaction mixtures. These products are a combination of A, C, G and T reaction products for several sequencing reactions. The products of the different sequencing reactions are labeled with fluorescent tags which are distinguishable one from the other on the basis of their excitation or emission spectra. After separation of the oligonucleotides by electrophoresis, the order of the detected peaks is used to call the base sequence.

BACKGROUND OF THE INVENTION

[0001] This application relates to an improved method for sequencing ofnucleic acids using multiple fluorescent labels, and to apparatus andkits adapted for use with the method.

[0002] Sequencing of nucleic acids using the chain termination methodinvolves the general steps of combining the target nucleic acid polymerto be sequenced with a sequencing primer which hybridizes with thetarget nucleic acid polymer; extending the sequencing primer in thepresence of normal nucleotide (A, C, G, and T) and a chain-terminatingnucleotide, such as a dideoxynucleotide, which prevents furtherextension of the primer once incorporated; and analyzing the product forthe length of the extended fragments obtained. Analysis of fragments maybe done by electrophoresis, for example on a polyacrylamide gel.

[0003] Although this type of analysis was originally performed usingradiolabeled fragments which were detected by autoradiography afterseparation, modern automated DNA sequencers generally are designed foruse with sequencing fragments having a fluorescent label. Thefluorescently labeled fragments are detected in real time as theymigrate past a detector.

[0004] U.S. Pat. No. 5,171,534 which is incorporated herein by referencedescribes a variation of this basic sequencing procedure in which fourdifferent fluorescent labels are employed, one for each sequencingreaction. The fragments developed in the A, G, C and T sequencingreactions are then recombined and introduced together onto a separationmatrix. A system of optical filters is used to individually detect thefluorophores as they pass the detector. This allows the throughput of asequencing apparatus to be increased by a factor of four, since the foursequencing reaction which were previously run in four separate lanes orcapillaries can now be run in one.

[0005] It is an object of the present invention to provide a furtherimprovement for use with chain termination sequencing reactions whichcan further increase the throughput of an instrument.

SUMMARY OF THE INVENTION

[0006] In order to use nucleic acid sequencing as a diagnostic tool, itwill be necessary to determine the sequence of the same DNA region frommany samples. The present invention makes it possible to increase thethroughput of an instrument being used for this purpose. Thus, a firstaspect of the invention provides a method for evaluating the sequence ofa target nucleic acid polymer in a plurality of samples. In this method,each sample is first divided into four aliquots which are combined withfour sequencing reaction mixtures. Each sequencing reaction mixturecontains a polymerase enzyme, a primer for hybridizing with the targetnucleic acid, nucleoside feedstocks and a different dideoxynucleoside.This results in the formation of an A-mixture, a C-mixture, a T-mixtureand a C-mixture for each sample containing product oligonucleotidefragments of varying lengths. The product oligonucleotide fragments arelabeled with fluorescent tags, and these tags will generally be the samefor all four sequencing reactions for a sample. However, the fluorescenttags used for each sample are distinguishable one from the other on thebasis of their excitation or emission spectra.

[0007] Next, the A-mixtures for each sample are combined to form acombined A mixture, the C-mixtures are combined to form a combinedG-mixture and so on for all four mixtures. The combined mixtures areloaded onto a separation matrix at separate loading sites and anelectric field is applied to cause the product oligonucleotide fragmentsto migrate within the separation matrix. The separated productoligonucleotide fragments having the different fluorescent tags aredetected as they migrate within the separation matrix.

[0008] The method of the invention can be used as described above todetermine the position of every base in the sequence, or it can be usedto determine the position of less than all four bases. For example, themethod can be used to determine the position of only the A bases withina sequence for some diagnostic applications.

[0009] A further aspect of the present invention is a kit useful fordiagnostic sequencing of a selected portion of a gene. One embodiment ofsuch a kit contains a plurality of sequencing primers for the selectedportion of the gene, each sequencing primer being identical in its DNAsequence but being labeled with a different fluorescent tag.

[0010] A further aspect of the invention is an apparatus for performingthe method of the invention. Such an apparatus comprises

[0011] (a) means for providing excitation energy to a detection sitewithin a separation matrix disposed within the apparatus;

[0012] (b) means for detecting light emitted from fluorescently-labeledoligonucleotide fragments located within the detection site;

[0013] (c) configuration control means, operatively connected to themeans for providing excitation energy and the means for detecting toprovide combinations of excitation wavelength and detection wavelengthspecific for a plurality of different fluorescently-labeledoligonucleotide fragments; and

[0014] (d) data processing means, operatively connected to theconfiguration control means and the means for detecting for receiving asignal from the means for detecting and assigning that signal to a datastream based upon the combination of excitation wavelength and detectionwavelength set by the configuration control means.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIGS. 1A and B shows a schematic representation of the method ofthe invention;

[0016]FIGS. 2A, B, C, and D show excitation and emission spectra fortheoretical sets of useful fluorescent tags; and

[0017]FIG. 3 shows an apparatus for evaluating the sequence of nucleicacid polymers using the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018]FIG. 1A shows a schematic representation of one embodiment of themethod of the invention. The figure depicts the application of themethod to two samples for clarity. As will be apparent from thediscussion below, however, the method of the invention is not limited totwo samples, and is in fact preferably applied for four or more samples,up to a limit imposed only by the number of distinguishable tags whichcan be identified.

[0019] As shown in FIG. 1A, two samples, “sample 1” and “sample 2” areeach divided into four aliquots and these aliquots are introduced intosequencing reactions A1, C1, G1, and T1, and A2, C2, G2 and T2. Eachsequencing reaction contains the reagents necessary for producingproduct oligonucleotide fragments of varying lengths indicative of theposition of one-base within the target nucleic acid sequence. Thesereagents include a polymerase enzyme, for example T7 polymerase,Sequenase™, Thermo Sequenase™, or the Klenow fragment of DNA polymerase;A, C, G and T nucleoside feedstocks; one type of chain terminatingdideoxynucleoside; and a sequencing primer.

[0020] After the product oligonucleotide fragments are formed in eachreaction mixture, the products from reaction mixture A1 are combinedwith the products from reaction mixture A2 to form a combined mixture 10which is loaded onto lane 1 of a separation matrix. Likewise, theproducts from reaction mixture C1 are combined with the products fromreaction mixture C2 to form a combined mixture 11 which is loaded ontolane 2 of the separation matrix; the products from reaction mixture C1are combined with the products from reaction mixture G2 to form acombined mixture 12 which is loaded onto lane 3 of the separationmatrix; and the products from reaction mixture T1 are combined with theproducts from reaction mixture T2 to form a combined mixture 13 which isloaded onto lane 4 of the separation matrix.

[0021] The key to the present invention is the use of labels in thereactions A1, C1, G1, and T1 which are distinguishable from the labelsused in reactions A2, C2, C2 and T2, respectively. Thus, unlike themethod described in U.S. Pat. No. 5,171,534 where the labels used forthe A, C, C, and T reactions for a sample are distinct, in the presentinvention the labels used for the four sequencing reactions for any onesample can be, and preferably are, the same. Instead, it is the labelswhich are used in the several samples which are distinct in the methodof the invention.

[0022] An alternative embodiment of the invention is illustrated in FIG.1B. In this case, the operator wants to sequence a plurality of genes(or different exons of the same gene) from one patient sample. Thesample 20 is divided into four aliquots. A sequencing reaction mixcontaining the reagents necessary for producing product oligonucleotidefragments of varying lengths is added to each aliquot. The sequencingmix added to a first aliquot contains all of the reagents for an Atermination reaction, plus a plurality of sequencing primers, each onelabeled with a distinguishable fluorophore, and each one being specificfor a different gene (or different exon of the same gene). Thesequencing mix added to a second aliquot contains all of the reagentsfor a C termination reaction, plus the same plurality of sequencingprimers. Sequencing reaction mixes for G and T are made in the samefashion. These sequencing mixture are reacted to produceoligonucleotides fragments, and then loaded onto lanes 21, 22, 23, and24 of a sequencing gel and separated. Using this technique, any numberof genes or exons in a sample can be simultaneously sequenced up to thelimit imposed by the number distinguishable tags which can beidentified.

[0023] Suitable labels for use in the present invention are fluorescenttags. These can be incorporated into the product oligonucleotidefragments in any way, including the use of fluorescently tagged primersor fluorescently tagged chain terminating reagents.

[0024] The fluorescent tags selected for use in the present inventionmust be distinguishable one from another based on their excitationand/or emission spectra. For example, as shown in FIG. 2A, a set of tagscould be selected which had overlapping emission spectra (Em1, Em2, Em3and Em4) but separate and distinguishable excitation spectra (Exl, Ex2,Ex3, and Ex4). A set of tags could also be selected which hadoverlapping excitation spectra but separate and distinguishable emissionspectra as shown in FIG. 2B. Further, as shown in FIG. 2C, a set of tagscould be selected in which some of the tags have overlapping excitationspectra (Ex1 and Ex2) but separate and distinguishable emission spectra(Em1 is distinguishable from Em2); while the others have separate anddistinguishable excitation spectra (Ex1, Ex3, and Ex4) but overlappingemission spectra (Em1, Em3 and Em4). A further combination of excitationand emission spectra is shown in FIG. 2D.

[0025] Examples of sets of suitable tags, together with the wavelengthmaximum for the excitation and emission spectra are shown in Table 1.Many other fluorophores are available that can be used as labels for DNAsequencing reaction products. Such dyes are available from AppliedBiosystems, Inc. (Foster City, Calif.), Molecular Probes, Inc. (Oregon)and others. TABLE 1 Fluorescent Dye's suitable for use with theinvention Excitation Fluorescent Dye Max (nm) Emission Max (nm) TexasRed X 599 617 Carboxy-X-Rhodamine 585 612 CarboxyFluorescein 494 521CarboxyTetraMethylRhodamine 561 591 Carboxycyanine 5.0 650 667

[0026]FIG. 3 shows a basic layout for an apparatus for evaluating thesequence of nucleic acid polymers using the method of the invention.Light from a light source 31, which may be for example a laser, a lightemitting diode, a laser diode, an incandescent or polychromatic lamp, orany combination of such sources, is passed through an optical filter 32to select an appropriate excitation wavelength which is directed to adetection site 33 in a separation matrix 34. Light emitted byfluorescent tags in the detection site 33 passes through a secondoptical filter 35 to a detector 36. Either or both of the opticalfilters 32 and 36 may be adjustable under the control of amicroprocessor, minicomputer or personal computer 37 to provide variousconfigurations of excitation and emission wavelengths as discussed morefully below. The output from the detector is then transmitted to a dataprocessing system such as a dedicated microprocessor, minicomputer orpersonal computer 37 for analysis to produce a report on the sequence ofthe sample being evaluated.

[0027] In the case where the properties of the selected tags are of thetype shown in FIG. 2A, the optical filter 32 may adjustable, for exampleby rotating several different filters through the path of the excitationbeam, to produce excitation beams corresponding to the differentexcitation wavelengths of the tags. Optical filter 35 may then be simplya cut-off filter selected to exclude light of the excitation wavelengthsfrom the detector. Information concerning the position of the opticalfilter 32 as a function of time is transmitted to the data processingsystem, and used to permit interpretation of the fluorescence data.Thus, when the optical filter 32 is in a position that corresponds tothe excitation spectrum of the tag used to label sample 1, the dataprocessing system interprets the emission intensity as data for thesequence of sample 1; when the optical filter 32 is in a position thatcorresponds to the excitation spectrum of the tag used to label sample2, the data processing system interprets the emission intensity as datafor the sequence of sample 2 and so on for as many different tags areused.

[0028] In the case where the properties of the selected tags are of thetype shown in FIG. 2B, the optical filter 35 is adjustable, for exampleby rotating several different filters through the path of the excitationbeam, to selectively collect emissions wavelengths corresponding to thedifferent tags. Optical filter 32 may be simply a cut-off or band-passfilter selected to exclude light of the emission wavelengths from thedetector. Information concerning the position of the optical filter 35as a function of time is transmitted to the data processing system, andused to permit interpretation of the fluorescence data. Thus, when theoptical filter 35 is in a position that corresponds to the emissionspectrum of the tag used to label sample 1, the data processing systeminterprets the emission intensity as data for the sequence of sample 1;when the optical filter 35 is in a position that corresponds to theemission spectrum of the tag used to label sample 2, the data processingsystem interprets the emission intensity as data for the sequence ofsample 2 and so on for as many different tags are used.

[0029] Finally, in the case where the properties of the selected tagsare of the type shown in FIG. 2C, both optical filter 32 and optical 35are adjustable in synchronization to control the excitation and emissionwavelengths being monitored. Information concerning the position of theoptical filters 32 and 35 as a function of time is transmitted to thedata processing system, and used to permit interpretation of thefluorescence data. Thus, when optical filter 32 is in a position thatcorresponds to excitation spectrum Ex1 in FIG. 2C, and optical filter 35is in a position that transmits the light of the wavelength of emissionspectrum Em1, the data processing system interprets the emissionintensity as data for the sequence of the sample labeled with tag 1.When the optical filter 35 is in a position to transmit light of thewavelength of emission spectrum Em2, on the other hand, the dataprocessing system interprets the emission intensity as data for thesequence of the of sample labeled with tag 2.

[0030] The light source 31 may be a single light source which is movedacross the gel to irradiate each detection zone individually, forexample as described in U.S. Pat. No. 5,207,880 which is incorporatedherein by reference. The light source 31 may also be a singe lightsource which is split into multiple beamlets, for example using opticalfibers or through the use of a beam splitter such as a spot arraygeneration grating to each of the detection sites as described generallyin U.S. patent application Ser. No. 08/353,932 and PCT PatentApplication No. PCT/US95/15951 which are incorporated herein byreference. The light source 31 may also be multiple individual lightsources, each of which irradiates a subset of one or more of thedetection sites within the separation matrix.

[0031] While optical filter 32 described above can be used effectivelyto provide selection of excitation wavelength, it will be understoodthat other approaches to providing different excitation wavelengths canbe used as well. For example a plurality of lasers of differentwavelengths could be used, with the light from each directed in turn tothe detection site. For detection of product oligonucleotide fragmentsin multiple lanes of an electrophoresis gel, one set of lasers might beused (one for each necessary wavelength) with light from each of thelasers being conducted by optical fibers or through the use of a beamsplitter such as a spot array generation grating to each of thedetection sites as described generally in U.S. patent application Ser.No. 08/353,932 and PCT Patent Application No. PCT/US95/15951. Opticalswitches, operatively connected to the data processing system, are usedto select which of the excitation wavelengths is striking the detectionzone at any given time, in the same manner as a rotating optical filtercould do. A detector assembly which scans across the lanes of anelectrophoresis gel could also be used, for example as described in U.S.Pat. Nos. 5,360,523 and 5,100,529, which are incorporated herein byreference, although the time required for such a device to scan all thelanes of a gel may be a limiting factor in applications with short totalmigration times.

[0032] It will also be appreciated that multiple optical filters mountedon individual detectors could be used in place of the adjustable opticalfilter 35 and the single detector 36 shown in FIG. 3. Similarly severaldetectors with adjustable optical filters might also be used.

[0033] Other optical components which separate light by wavelength mayalso be used in place of optical filter 35. Thus, for example, adiffraction grating or prism which spatially separates light ofdiffering wavelength may be employed. In this case, radiation from thedifferent fluorophores will be distributed in space, and can be detectedby dedicated detectors such as photodiodes, CCD elements (linear orX-Y). Similarly, distinct optical filters for each wavelength can beemployed in combination with a multiplicity of detectors. Opticalfilters which are transmissive at the emission wavelength of onefluorophore and reflective at the emission wavelength of a secondfluorophore can also be used to direct emitted light to separatedetectors depending on wavelength.

[0034] The detectors used in the apparatus of the invention may bephotomultipliers, photodiodes or a CCD. As noted above, the apparatuscan be configured with one or more detectors for each detection site.The apparatus can also be configured such that one detector overlapswith several detection sites if the excitation light is directed to thedetection sites one at a time. In addition, the apparatus can use asingle detector (or a small array of detectors) which is scanned acrossthe gel during detection.

[0035] For determining the sequence of a selected region of DNA usingthe method of the invention, a kit may be formulated. Such a kitcomprises, in packaged combination, a plurality of containers, eachcontaining a reagent for the sequencing of the selected region of DNA.The reagent in each container has a reactive portion, which is involvedin the sequencing reaction, and a fluorescent label portion. The labelportions of the reagents in each container are different anddistinguishable one from the other on the basis of the excitation oremission spectra thereof.

[0036] In a preferred embodiment, a kit in accordance with the inventioncomprises a plurality of primers for sequencing the selected region,each of the primers having a different and distinguishable fluorescentlabel. Thus, the reactive portions of the reagents in this case are theoligonucleotide primer to which the labels are attached. The reactiveportions may be different from one another, but are preferably the same.Such a kit may also include additional reagents for sequencing,including polymerase enzymes, dideoxynucleosides and buffers.

[0037] Alternatively, the kit may contain one primer for the selectedregion and a plurality of containers of chain-terminating nucleosides,each labeled with a different and distinguishable fluorescent label. Inthis case, the reactive portion of the reagent is the chain terminatingnucleoside which can be incorporated in place of a normal nucleosideduring the sequencing reaction. The kit may include reagents having justone type of chain-terminating nucleoside, for example ddA with aplurality of distinct fluorescent labels, or it may include reagentshaving two or more types of chain-terminating nucleosides, each with aplurality of distinct labels. Such a kit may also include additionalreagents for sequencing, including polymerase enzymes and buffers, aswell as additional chain-terminating nucleosides (single-labeled) forthose not provided as part of a multi-label reagent set.

[0038] For practising the method shown in FIG. 1B, a suitable kit inaccordance with the invention includes at least one container containinga mixture of a plurality of sequencing primers, one for each gene regionto be evaluated. The plurality of sequencing primers each comprise areactive portion which hybridizes with DNA in the sample and a labelportion, the label portions of the reagents being different anddistinguishable one from the other. Preferably, the detectable labelsare fluorescent tags, distinguishable one from the other by theiremission or excitation spectra.

What is claimed is:
 1. A method for evaluating the nucleic acid sequenceof a plurality of samples comprising the steps of (a) obtaining a firstaliquot of each sample; (b) combining the first aliquot of each samplewith a sequencing reaction mixture containing a polymerase enzyme, aprimer for hybridizing with the sample, nucleoside feedstocks and afirst dideoxynucleoside to form a first plurality of mixtures of productoligonucleotide fragments, one for each sample, wherein the productoligonucleotide fragments formed from each first aliquot are labeledwith different fluorescent tags, said different fluorescent tags beingdistinguishable one from the other on the basis of their excitation oremission spectra; (c) combining the first plurality of mixtures ofoligonucleotide products to form a first combined mixture; (d) loadingthe first combined mixture onto a separation matrix at a first loadingsite; (e) applying an electric field to cause the oligonucleotideproducts to migrate within the separation matrix; and (f) detecting theoligonucleotide products having the different fluorescent tags as theymigrate within the separation matrix.
 2. The method according to claim1, further comprising the steps (i) obtaining a second aliquot of eachsample; (ii) combining the second aliquot of each sample with asequencing reaction mixture containing a polymerase enzyme, a primer forhybridizing with the sample, nucleoside feedstocks and a seconddideoxynucleoside different from the first dideoxynucleoside to form asecond plurality of mixtures of product oligonucleotide fragments, onefor each sample, wherein the product oligonucleotide fragments formedform each second aliquot are labeled with different fluorescent tags,said different fluorescent tags being distinguishable one from the otheron the basis of their excitation or emission spectra; (iii) combiningthe second plurality of mixtures of oligonucleotide products to form asecond combined mixture; (iv) loading the second combined mixture onto aseparation matrix at a second loading site, distinct from the firstloading site; (v) applying an electric field to cause theoligonucleotide products to migrate within the separation matrix; and(vi) detecting the oligonucleotide products having the differentfluorescent tags as they migrate within the separation matrix.
 3. Themethod according to claim 2, wherein the first and second aliquots arecombined with a sequencing reaction concurrently, and wherein the firstand second combined mixtures are loaded onto different lanes of the samegel.
 4. The method according to claim 3, wherein the same primer andfluorescent tag are combined with the first and second aliquots of eachsample.
 5. The method according to claim 1, wherein first aliquots fromat least three samples are combined to form the first combined mixture.6. The method according to claim 1, wherein first aliquots from morethan four samples are combined to form the first combined mixture.
 7. Amethod for evaluating the sequence of a target nucleic acid polymer in aplurality of samples comprising the steps of (a) obtaining four aliquotsof each sample; (b) combining the aliquots of each sample with foursequencing reaction mixtures, each sequencing reaction mixturecontaining a polymerase enzyme, a primer for hybridizing with the targetnucleic acid, nucleoside feedstocks and a different dideoxynucleoside toform an A-mixture, a G-mixture, a T-mixture and a C-mixture for eachsample containing product oligonucleotide fragments of varying lengths,wherein the product oligonucleotide fragments are labeled withfluorescent tags, and the fluorescent tags used for each sample aredistinguishable one from the other on the basis of their excitation oremission spectra; (c) combining the A-mixtures, the C-mixtures, theT-mixtures and the C-mixtures for each sample to form a combinedA-mixture, a combined C-mixture, a combined t-mixture and a combinedC-mixture; (d) loading the combined A-mixture, the combined G-mixture,the combined T-mixture and the combined C-mixture onto a separationmatrix at separate loading sites; (e) applying an electric field tocause the product oligonucleotide fragments to migrate within theseparation matrix; and (f) detecting the product oligonucleotidefragments having the different fluorescent tags as they migrate withinthe separation matrix.
 8. An apparatus for evaluating the sequence of anucleic acid polymer by separation of a reaction mixture containingoligonucleotide fragments in a separation matrix disposed within theapparatus comprising (a) means for providing excitation energy to adetection site within the separation matrix; (b) means for detectinglight emitted from fluorescently-labeled oligonucleotide fragmentslocated within the detection zone; (c) configuration control means,operatively connected to the means for providing excitation energy andthe means for detecting to provide combinations of excitation wavelengthand detection wavelength specific for a plurality of differentfluorescently-labeled oligonucleotide fragments; and (d) data processingmeans, operatively connected to the configuration control means and themeans for detecting for receiving a signal from the means for detectingand assigning that signal to a data stream based upon the combination ofexcitation wavelength and detection wavelength set by the configurationcontrol means.
 9. A kit for determining the sequence of a selectedregion of DNA comprising, in packaged combination, at least of first setof a plurality of containers, each container containing a reagentspecies for the sequencing of the selected region of DNA, wherein thereagent species in each container comprises a reactive portion and alabel portion and wherein the label portions of the reagents aredifferent and distinguishable one from the other.
 10. The kit accordingto claim 9, wherein the detectable labels are fluorescent tags,distinguishable on the basis of the excitation or emission spectrathereof.
 11. The kit according to claim 10, wherein the reactive portionof each reagent species in the first set of containers is anoligonucleotide primer for use in sequencing the selected region of DNA.12. The kit according to claim 11, wherein the reactive portions of eachreagent species in the first set of containers are the same as oneanother.
 13. The kit according to claim 11, wherein the reactive portionof each reagent species in the first set of containers is adideoxynucleoside and wherein the reactive portions of each reagentspecies in the first set of containers are the same as one another. 14.The kit according to claim 13, further comprising a second set ofcontainers, each containing a reagent species for the sequencing of theselected region of DNA, wherein the reagent species in each container ofthe second set comprises a dideoxynucleoside reactive portion differentfrom the reactive portion of the reagent species in the first set ofcontainers, and a fluorescent label portion and wherein the labelportions of the reagents in the second set of containers are differentand distinguishable one from the other on the basis of the excitation oremission spectra thereof.
 15. A method for evaluating the sequence of aplurality of gene regions within a sample comprising the steps of: (a)combining at least a first aliquot of the sample with a sequencingreaction mixture containing a polymerase enzyme, a plurality ofsequencing primer species, one sequencing primer species for each generegion, nucleoside feedstocks and a first dideoxynucleoside to form afirst mixture of product oligonucleotide fragments, wherein each of thesequencing primer species is labeled with a different detectable label,said different detectable labels being distinguishable one from theother by a detection system; (b) separating the first mixture of productoligonucleotide fragments based upon the size of the fragments; (c)detecting emissions from the separated oligonucleotide fragments foreach different detectable label; and (d) evaluating the sequence of eachgene region based upon the oligonucleotide fragments detected.
 16. Themethod according to claim 15, wherein the detectable labels arefluorescent tags.
 17. The method according to claim 16, whereinfluorescent tags are distinguishable based upon their excitation oremission spectra.
 18. The method according to claim 15, wherein fouraliquots of sample are combined with four separate sequencing reactionmixtures, each containing a different dideoxynucleoside.
 19. A kit forevaluating the sequence of a plurality of gene regions within a samplecomprising, in packaged combination, at least one container containing amixture of a plurality of sequencing primers, one for each gene regionto be evaluated, wherein the plurality of sequencing primers eachcomprise a reactive portion which hybridizes with DNA in the sample anda label portion and wherein the label portions of the reagents aredifferent and distinguishable one from the other.
 20. The kit accordingto claim 20, wherein the detectable labels are fluorescent tags,distinguishable one from the other by their emission or excitationspectra.